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Chapter 3.8 : Fecal and Other Gastrointestinal Cultures and Toxin Assays

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Abstract:

Gastroenteritis can be caused by bacteria, parasites, or viruses. With such a wide array of pathogens and the need for cost containment, physician input and practice guidelines ( ) can help the laboratory determine which tests are appropriate for detecting the etiological agent of diarrhea. Microbiology laboratories should review the local epidemiology of bacterial enterocolitis and implement routine stool culture methods that will allow recovery and detection of all of the major pathogens causing most of the cases in their geographic area. All microbiology laboratories should routinely test for the presence of spp., spp., and spp. on all stool cultures. Other major pathogens, such as Shiga-toxin-producing , particularly O157 or enterohemorrhagic (EHEC), should also be routinely tested for on bloody stool samples during the spring, summer, and early fall months in geographic areas where the prevalence of these strains has been shown to be increased. Microbiology laboratories situated in or near coastal communities may also test for and spp. since the prevalence of these types of infections is increased with exposure to water or contaminated food such as shellfish.

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
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Image of Figure 3.8.1-1
Figure 3.8.1-1

Flowchart for the identification of oxidase-positive stool pathogens from BAP or from either TCBS or CIN. Most are also indole positive. Biochemical reactions for species identification are available on many commercial kits. Growth on TCBS implies that the organism is a sp., but not all spp. grow on TCBS. Abbreviations: MH, Mueller-Hinton agar; ID, identification; K, alkaline; A, acid; r/o, rule out.

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
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Image of Figure 3.8.1-2
Figure 3.8.1-2

Flowchart for identification of stool pathogens from routine stool cultures. Set up either TSI or KIA, BAP, and urea agar (or rapid urea tube) from all lactosenegative or HS-positive colonies on enteric selective agars. Reactions of the slant are listed with a slash before the butt reaction. Optionally for HS-negative colonies, Andrade's glucose tube with Durham tube for gas will eliminate most questionable production of gas and provide a broth for VP testing. Perform spot tests (indole, oxidase, PYR) only from BAP. r/o, rule out.

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
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Image of Figure 3.8.2–1
Figure 3.8.2–1

identification flowchart for minimum identification of from stool specimens. Abbreviations: R, no zone; S, zone.

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
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Image of Figure 3.8.3-1
Figure 3.8.3-1

Example of worksheet diagram to accompany a 96-well microtiter tray previously inoculated with a monolayer of fibroblasts and subsequently inoculated with patient specimen, controls, toxin, and antitoxin. Day 1 is the first date the tray is used. Day 2 illustrates the controls omitted on subsequent days; i.e., MEM and antitoxin control wells are not needed. Patient stool specimens in dilutions of 1:20 and 1:100 are indicated as patient A, B, C, D, and E, inoculated on day 1, and patients F and G, inoculated on day 2. Note that outer wells are not used. A, antitoxin.

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
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8. Laine, L.,, R. Estrada,, D. N. Lewin,, and H. Cohen. 1996. The influence of warming on rapid urease test results—a prospective evaluation. Gastrointest. Endosc. 44: 429 432.
9. Laine, L.,, D. Lewin,, W. Naritoku,, R. Estrada,, and H. Cohen. 1996. Prospective comparison of commercially available rapid urease tests for the diagnosis of Helicobacter pylori. Gastrointest. Endosc. 44: 523 526.
10. Leung, W. K.,, and D. Y. Graham. 2000. Clarithromycin for Helicobacter pylori infection. Exp. Opin. Pharmacother. 1: 507 514.
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12. Piccolomini, R.,, G. Di Bonaventura,, D. Festi,, G. Catamo,, F. Laterza,, and M. Neri. 1997. Optimal combination of media for primary isolation of Helicobacter pylori from gastric biopsy specimens. J. Clin. Microbiol. 35: 1541 1544.
13. Vaira, D.,, P. Malfertheiner,, F. Mégraud,, A. T. R. Axon,, M. Deltenre,, A. M. Hirschl,, G. Gasbarrini,, C. O'Morain,, J. M. Pajares Garcia,, M. Quina,, G. N. J. Tytgat, and the HpSA European Study Group. 1999. Diagnosis of Helicobacter pylori infection with a new non-invasive antigen-based assay. Lancet 354: 30 33.
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1. Evangelista, A. T.,, A. L. Truant,, and P. Bourbeau,. 2002. Rapid systems and instruments for the identification of bacteria, p. 38 39. In A. L. Truant (ed.), Manual of Commercial Methods in Microbiology. ASM Press, Washington, DC.
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3. Vaira, D.,, P. Malfertheiner,, F. Me`graud,, A. Axon,, M. Deltenre,, A. M. Hirschl,, G. Gasbarrini,, C. O'Morain,, J. M. Pajares,, M. Quina,, G. N. J. Tytgat, and the HpSA European Study Group. 1999. Diagnosis of Helicobacter pylori infection with a new noninvasive antigen-based assay. Lancet 354: 30 33.
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1.Centers for Disease Control and Prevention. 1994. Preventing the spread of vancomycin resistance—report from the Hospital Infection Control Practices Advisory Committee. Fed. .Regist. 59: 2575825763.
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3. Clinical and Laboratory Standards Institute. 2006. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 7th ed. Approved standard M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA.
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8. Ieven, M.,, E. Vercauteren,, P. Descheemaeker,, F. van Laer,, and H. Goossens. 1999. Comparison of direct plating and broth enrichment culture for the detection of intestinal colonization by glycopeptide-resistant enterococci among hospitalized patients. J. Clin. Microbiol. 37: 1436 1440.
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10. Ledeboer, N. A.,, K. Das,, M. Eveland,, C. Roger-Dalbert,, S. Mailler,, S. Chatellier,, and W. M. Dunne. 2007. Evaluation of a novel chromogenic agar medium for isolation and differentiation of vancomycin-resistant Enterococcus sfaecium mand Enterococcus sfae-calis. J. .Clin. .Microbiol. 44: 4561 4563.
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Tables

Generic image for table
Table 3.8.1-1

Commonly used primary plating and broth media for isolation of and

Either bile salts, deoxycholate, or Selenite is present in each medium to inhibit gram-positive microbiota. Abbreviations: D, differential; E, enriched; S, selective. Ferric ammonium citrate reacts with hydrogen sulfide (HS) from organism to produce black color of colony.

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Table 3.8.1-2

Special highly selective media for specific pathogen requests

Incubate at 37°C in O unless otherwise stated.

Since as many as 20% of asymptomatic hospitalized patients may be colonized with , tests for the presence of toxin in stool are more specific for diagnosis of -associated diarrhea. Isolation of the organism should only be done for epidemiological studies, with confirmation that the isolated strain is a toxin producer. CCFA (containing cycloserine, cefoxitin, fructose, egg yolk, and neutral red) can also be used for isolation. Do not use a medium with neutral red to demonstrate colonial fluorescence ( ).

Prepare TCBS fresh for use from powder or by melting previously prepared or purchased “deeps.” If made from powder, boil but do not autoclave prior to use.

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Table 3.8.1-3a

QC of specialized media for detection of fecal pathogens

Abbreviations are as follows. A, for testing nutritive properties. Inoculate each medium with 10 µl of a 1:100 dilution of standardized cell suspension (0.5 McFarland). If isolated colonies are not obtained, use a 10-fold-lighter inoculum. B, for testing selective properties. Inoculate each medium with 10 µl of a 1:10 dilution of standardized cell suspension (0.5 McFarland). Although ATCC strains are listed, any organism that will yield the identical result is acceptable. For medium abbreviations, refer to Table 3.8.1-2 .

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Table 3.8.1-3b

QC of specialized media for detection of fecal pathogens

Abbreviations are as follows. A, for testing nutritive properties. Inoculate each medium with 10 µl of a 1:100 dilution of standardized cell suspension (0.5 McFarland). If isolated colonies are not obtained, use a 10-fold-lighter inoculum. B, for testing selective properties. Inoculate each medium with 10 µl of a 1:10 dilution of standardized cell suspension (0.5 McFarland). Although ATCC strains are listed, any organism that will yield the identical result is acceptable. For medium abbreviations, refer to Table 3.8.1-2 .

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Table 3.8.1-4

Microscopic and gross observations of fecal specimens associated with various infections

Data are only a guideline, and in any infection, observations are variable. For example, only 50% of -associated cases of diarrhea demonstrate the presence of PMNs.

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Table 3.8.1-5

Biochemical differentiation of selected members of the group

Symbols: −, =9% of strains positive; V, 10 to 89% of strains positive; +, ≥90% of strains positive.

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Table 3.8.1-6

Summary of detection media and identification methods for fecal pathogens

For species, see procedure 3.8.2. NA, not applicable.

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Table 3.8.2–1a

Taxonomic position, known sources, and common disease associations of , , , and related species

able adapted from reference .

All organisms listed belong to rRNA superfamily VI. Organisms in boldface type are associated wih human sources. Validly published or most commonly used nomenclature of the taxa described are given priority, with superseded (in quotation marks) or less common nomenclature given in parentheses. Names that have not been validly published are given in single quotation marks. Abbreviations: CNW, catalase negative—weak; NNC, nitrate-negative ; NARTC, NA-resistant thermophilic ; CLO, -like organism; IDO, intracellular organism.

Likely phylogenetic position of taxon in rRNA superfamily VI based on 16S rRNA sequence comparisons or DNA-DNA hybridization data.

Biovar descriptions conform with recent suggestions by On ( ).

Original phylogenetic position in rRNA superfamily VI emended with reference to available 16S rRNA sequence comparisons. Brackets indicate that the taxon is generically misnamed.

The proposed name of ‘’ did not distinguish between the two phylogenetically distinct taxa referred to as ‘ 1’ and ‘ 2.’

Taxonomic position based upon marked morphological similarity to ‘’ spp.

Most current taxonomy indicates to be indistinguishable from ( ).

canadensis is a newly described agent of human gastroenteritis and is closely related to ( ).

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Table 3.8.2–1b

Taxonomic position, known sources, and common disease associations of , , , and related species

able adapted from reference .

All organisms listed belong to rRNA superfamily VI. Organisms in boldface type are associated wih human sources. Validly published or most commonly used nomenclature of the taxa described are given priority, with superseded (in quotation marks) or less common nomenclature given in parentheses. Names that have not been validly published are given in single quotation marks. Abbreviations: CNW, catalase negative—weak; NNC, nitrate-negative ; NARTC, NA-resistant thermophilic ; CLO, -like organism; IDO, intracellular organism.

Likely phylogenetic position of taxon in rRNA superfamily VI based on 16S rRNA sequence comparisons or DNA-DNA hybridization data.

Biovar descriptions conform with recent suggestions by On ( ).

Original phylogenetic position in rRNA superfamily VI emended with reference to available 16S rRNA sequence comparisons. Brackets indicate that the taxon is generically misnamed.

The proposed name of ‘’ did not distinguish between the two phylogenetically distinct taxa referred to as ‘ 1’ and ‘ 2.’

Taxonomic position based upon marked morphological similarity to ‘’ spp.

Most current taxonomy indicates to be indistinguishable from ( ).

canadensis is a newly described agent of human gastroenteritis and is closely related to ( ).

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Table 3.8.2–1c

Taxonomic position, known sources, and common disease associations of , , , and related species

able adapted from reference .

All organisms listed belong to rRNA superfamily VI. Organisms in boldface type are associated wih human sources. Validly published or most commonly used nomenclature of the taxa described are given priority, with superseded (in quotation marks) or less common nomenclature given in parentheses. Names that have not been validly published are given in single quotation marks. Abbreviations: CNW, catalase negative—weak; NNC, nitrate-negative ; NARTC, NA-resistant thermophilic ; CLO, -like organism; IDO, intracellular organism.

Likely phylogenetic position of taxon in rRNA superfamily VI based on 16S rRNA sequence comparisons or DNA-DNA hybridization data.

Biovar descriptions conform with recent suggestions by On ( ).

Original phylogenetic position in rRNA superfamily VI emended with reference to available 16S rRNA sequence comparisons. Brackets indicate that the taxon is generically misnamed.

The proposed name of ‘’ did not distinguish between the two phylogenetically distinct taxa referred to as ‘ 1’ and ‘ 2.’

Taxonomic position based upon marked morphological similarity to ‘’ spp.

Most current taxonomy indicates to be indistinguishable from ( ).

canadensis is a newly described agent of human gastroenteritis and is closely related to ( ).

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Table 3.8.2–2

Human disease associations of species by clinical syndrome

GI, gastrointestinal.

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Table 3.8.2–3

Commercial systems for generating microaerobic environments and the approximate atmospheric content

This system produces negligible H and may not grow H-requiring species.

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Table 3.8.2–4

Phenotypic reactions of clinically important and species

+, positive reaction; 0, negative reaction; w, weakly positive; V, variable reaction, NA, not available. See procedure 3.8.4 for identification.

Urease-positive thermophilic campylobacters or -like strains may be found ( ).

Growth at 42°C; catalase negativity suggests .

/CLO1B can be separated by DNA homology tests. /CLO1B, , and can be definitively identified by cellular fatty acid analysis ( ).

Rare subsp. fetus strains are aerobic.

These species are historically sensitive to NA; however, resistant strains are seen in as high as 35% of isolates due to acquired fluoroquinolone resistance, which may make this assay less useful in identification.

HS in TSI suggests .

There are isolated reports of species that are urease producing other than ( ).

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Untitled

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Untitled

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Untitled

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8
Generic image for table
Untitled

Citation: Garcia L. 2010. Fecal and Other Gastrointestinal Cultures and Toxin Assays, p 209-268. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch3.8

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